Automated intracellular manipulation and transfection

ABSTRACT

A method and apparatus whereby prokaryotic, eukaryotic and/or mammalian cells may have genetic agents inserted or removed or transferred in an automated and semi-quantitative fashion. The functional unit of the invention is composed of two rectangular plates: a contact plate with circular holes and a carefully aligned base plate with a pleurality of rods, which protrude through the center of the holes of the contact plate. The edges of the two plates are sealed lengthwise to create a space whereby fluid may flow through the proximal and distal openings to induce a negative pressure, vacuum suction, venturi effect at each hole of the contact plate. The rods and base plate may be coated with an electronically magnetizable surface which may attract and hold or repulse and release any magnetically responsive genetic agents.

BACKGROUND

Somatic nuclear cell transfer is the process of removing the nucleus ofa cell and then transplanting it into an empty donor cell. This processhas been utilized for mammalian cloning in animal husbandry.Additionally, the procedure has been considered for use in limitedtherapeutic cloning, in which donor nuclei are transferred intopleuripotent enucleated surrogate cells in an autologous, allogenic orxenographic fashion.

Typically expensive and sensitive micromanipulation equipment is used toperform the procedure under direct microscopic visualization with atleast two micromanipulation armatures: one to hold and position the celland the other to lance the cell and introduce the transferred nucleus.Those skilled in the art of embryology and cellular biology are awarethat current equipment and technical skill required to perform nuclearcell transfer are prohibitively expensive, tedious and time consuming.

Transient transfection is also another challenging procedure in cellularand molecular biology. Many different types of genetic manipulatingagents may be physically introduced into the cell with direct injectionusing ballistics, electroporation or transfection agents such aslipofectamine.

For the purposes of this patent genetic agents may be referred to, butnot limited to, the following:

-   RNA (Ribonucleic Acid)-   DNA (Deoxiribonucleic Acid)-   PNA (Protien Nucleic Acid)-   siRNA (RNA intereference agents)-   whole chromosomes-   partial chromosomes-   artificial chromosomes-   protein-   enzymes-   oligonucleotides-   antibodies-   oligonucleotides-   signal transduction agents-   toxins-   hormones-   small molecules-   isotopes-   cell nuclei-   gametes-   or any other material that may induce a change in cellular activity    at the DNA, RNA, protein and/or signal transduction level.

For the purposes of this patent labels may be referred to, but notlimited to, the following:

-   antibodies-   synthetic oligonucleotides-   radio-isotopes-   DNA-   RNA-   siRNA-   PNA-   LNA (locked nucleic acid)-   nucleic acids-   amino acids-   protein-   haptens-   prions

Transfection using any of the above methods involves extensivemanipulation to the cell causing considerable loss in viability.Furthermore, trying to achieve direct quantitative transfection of thegenetic manipulating agents is not possible with the above methods.

Yet another challenging field of molecular diagnosis is capture ofnuclear proteins. Several nuclear proteins are now used as potentialmarkers for clinical utility. Rapid isolation and diagnosis of thepresence of these proteins is time consuming and challenging sometimesdue to the harsh denaturing chemical treatments necessary for nuclearprotein processing, which destroy delectability of nuclear targets.

Certainly an automated and inexpensive method with the versatility ofbeing capable of performing all the cellular and molecular proceduresdetailed previously would have considerable utility and biomedicalapplication.

SUMMARY OF INVENTION

Those familiar with the field of embryology and somatic nuclear celltransfer are aware that a donor nucleus must be removed from a donorcell and transferred into an empty surrogate cell. Essentially, thesurrogate cell must be cleared of pre-existing genetic material, namelythe nucleus prior to having the donor nucleus transplanted into thesurrogate cell. With current technology the process is performed bymicromanipulation devices composed of a vacuum pipette tip to hold thesurrogate cell and a glass pipette tip to lance the surrogate cell andinject the nucleus into the surrogate cell. The same procedure may beperformed by the invention disclosed such that conceptually the lancingelement and vacuum pipette is combined into one functional unit.

In simplest terms the functional unit of the patent is composed of tworectangular plates: a contact plate with circular and/or fruste-conicaledged holes and a carefully aligned base plate with a pleurality ofrods, which protrude through the holes of the contact plate and abovethe plane of the upper contact plate. The edges of the two plates aresealed lengthwise to create a space whereby fluid may flow between theplates and through the proximal and distal openings to induce a negativepressure, vacuum suction, or venturi force at each hole of the contactplate in a direction perpendicular and toward the plane of the baseplate. The proximal and distal openings may also have adjustable flowports to modulate the flow rate of fluid through the device andeffectively change the magnitude of negative pressure, vacuum suction orventuri effect at each hole. The distal opening may also be completelyoccluded to cause a positive pressure flowing away from the base platethrough the holes in the contact plate to push off any cell that waspreviously seated on the hole. The entire device and process may becarried out within a Petri dish and/or under a microscope. The rods andbase plate may be coated with an electronically magnetizable surface. Inthe ideal embodiment the fruste-conical holes of the base plate areoriented towards the cells to allow a spheroid-type shaped cell to seatitself securely over the hole when a negative pressure is appliedthrough the hole.

For the apparatus to be used for nuclear cell transfer, prior to anycontact with the apparatus, donor cells are incubated with magnetizedantibodies to the nucleus. After the donor nucleus is significantlycoated with magnetic antibodies the donor cells are introduced to theapparatus chamber, wherein they are attracted to the functional unit bya negative pressure vacuum. As the cell is pulled down and seats intothe fruste-conical base of the functional unit, the central pole of apiercing rod enters into the cell. The piercing rod is then magnetizedand the nucleus is captured on the rod. The negative pressure on theapparatus is reversed such that the donor cell is pushed of the baseplate leaving the donor nucleus behind and magnetically immobilizedagainst the piercing rod. The upper chamber and surface of the contactplate is then thoroughly washed so that no residual donor cell cytoplasmor membranes are left behind. Next the enucleated surrogate cells areintroduced to the surface of the contact plate. (Note that the surrogatecells may be pretreated in a similar manner, but performed within asimilar and separate apparatus to enucleate the cells' nuclei.) Thenegative vacuum flow is resumed and the surrogate cells are pulled ontothe functional unit and pierced by the magnetized rod holding theimmobilized donor nucleus. Once the donor cell has seated itself firmlyon the rod, the magnetic field of the rod is turned off or polarityreversed to release or repulse the donor nucleus into the cytoplasm ofthe surrogate cell. The surrogate cell is then released from the base ofthe base plate by reversing the negative pressure to push off thesurrogate cell now with the donor nucleus within it.

Alternatively the same apparatus may be used for transfection orinjection of other genetic agents. The procedure is performed in mannersimilar to somatic nuclear transfer, but instead or donor nuclei beingtransferred, other magnetized genetic agents are introduced into thesurrogate cells. Additionally, the surrogate cells in this alternateprocess may in fact retain its original nucleus. The magnetic field ofthe apparatus' magnetizable rods are turned on and attract themagnetized genetic agents, essentially coating the rod. The recipientcell is then introduced to the apparatus chamber and upper surface ofthe contact plate, and the recipient cells are then seated onto theholes of the contact plate. The magnetic field of the magnetizable rodsare then turned off or polarity reversed and the magnetized geneticagent is released or repulsed into the cytoplasm of the cell. The cellsare then pushed off the base plate by reversing the flow though the holeto push off the cell. The cell is then transfected with the magnetizedgenetic agent. The functional unit may be composed of a pleurality ofmagnetizable rods per hole to provide quantitative or semi-quantitativeincrements of transfection.

Additionally, the device may also be used for intracytoplasmic sperminjection. Those familiar with the field of animal husbandry andinfertility are aware that sperm may be physically injected into embryowith the use of micromanipulators. The same procedure may also becarried out with the disclosed invention, such that the sperm are coatedwith any suitable magnetizable antibody and immobilized onto themagnetized rod of the apparatus. A one sperm to one rod arrangement isideal. The embryos are placed onto the contact plate and fluid flowthough the proximal and distal ends of the apparatus induce a negativepressure through the holes in a direction towards the base platecreating a mild suction to seat a single embryo over a single rod coatedwith a single sperm. The magnetic field of the base plate and rod isthen turned off and the flow though the distal end of the apparatus(exit port) is blocked to cause flow of fluid through the holes awayfrom the base plate to push off the embryos and complete theintracytoplasmic sperm injection process.

Furthermore, the device may also be used for nuclear moleculardiagnosis. Nuclei from cells of interest may be removed for directstaining or labeling with antibodies, FISH, PCR probes, syntheticoligonucleotides or any suitable probe that is visually detectable ormeasureable. The cells of interest would be prepared in a similar mannersuch that a magnetized antibody recognizing a nuclear structure isexposed to the cell. The cells are then introduced to the apparatus toallow one cell to be seated onto one rod-hole unit as mentionedpreviously by modifying fluid flow through the proximal and distal ends(entry and exit ports) of the apparatus. The rod and base plate is thenmagnetized to capture the nuclei and the cell membrane is pushed off therod by blocking flow through the distal end (exit port) of the apparatusto expose the nucleus. The nucleus may then undergo exposure to anysuitable molecular probe with greater ease, since there is no impedingcytoplasm to occlude binding or visualization of probes. Additionally,the nuclear proteins and structure are left intact since no harshdenaturing lysis buffers are required.

Furthermore, the invention may be constructed from any suitable materialand by any suitable means to achieve the functional capabilitiesoutlined above. Those familiar with the field of lithography ananotechnology are aware that the apparatus of the invention describedmay be fabricated at the microscopic and/or nanometric level with usingmany tools and methods commonly available in the field of micro and nanofabrication.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. A conceptual drawing of the smallest functional unit of theinvention.

FIG. 2: A conceptual drawing of the smallest functional unit of theinvention minus the central magnetic rod in the center. Black arrowrepresenting negative pressure through center of functional unitgenerated as a result of fluid flow between upper and lower platesrepresented by large white arrow.

FIG. 3: Conceptual drawing of smallest functional unit in an idealconfiguration with a eukaryotic cell.

FIG. 4: Close up view (dotted inset) of magnetized antibody binding toantigens on nuclei and magnetized antibodies in turn being attracted tothe magnetic rod. (Magnetic attraction only used for this embodiment.)

FIG. 5: Close up view (dotted box inset) of antibody binding to antigenson nuclei constant region of antibody bound to antibodies immobilized onthe magnetizable rod of the apparatus. (No magnetic antibody or magneticattraction used for this embodiment.)

FIG. 6: Close up view (dotted box inset) of antibody binding to antigenson nuclei constant region of antibody bound to antibodies immobilized onthe magnetizable rod of the apparatus in addition to using magneticantibodies to magnetize the nucleus and allow attraction to the magneticrod. (Combined use of direct antibody binding and magnetic attraction tomagnetizable rod.)

FIG. 7: Conceptual drawing of smallest functional unit after donor cellis pushed off of apparatus and donor nucleus is still left behind on themagnetized rod.

FIG. 8: Conceptual drawing of the smallest functional unit of theapparatus being prepared for intracytoplasmic sperm injectionapplication.

FIG. 9: Conceptual drawing of the smallest functional unit of theapparatus being used for intracytoplasmic sperm injection application.

FIG. 10: Conceptual drawing of the smallest functional unit of theapparatus being prepared for transfection of genetic agents.

FIG. 11: Conceptual drawing of the smallest functional unit of theapparatus being used for transfection of genetic agents.

FIG. 12: Alternate conceptual drawing of the apparatus modified fortransfection of genetic agents, but in this case three times the amountof transfection may occur as there are 3 rods.

FIG. 13: 3-fold transfection of genetic agents into the recipient cell.

FIG. 14: Alternate embodiment of genetic agent transfer. usingmagnetized antibodies to attract bound genetic agents to magnetizedmagnetizable rod.

FIG. 15: An ideal three dimensional representation of the invention.Note magnetizable rods fit through center of upper plate.

FIG. 16: Longitudinal cross section representation of device.

FIG. 17: Saggital cross section view of device.

FIG. 18: Three dimensional view of patent with entry and exit flowthrough ports.

DETAILED DESCRIPTION OF DRAWINGS

FIG. 1. A conceptual drawing of the smallest functional unit of theinvention, whereby the upper contact plate (1) has a central hole (3)through which a magnetizable rod (4) protrudes though and above thesurface plane of the upper contact plate (1) and the fruste-conical edge(3). The magnetizable rod (4) is physically attached to the base plate(5) and both may also be electronically magnetizable.

FIG. 2: A conceptual drawing of the smallest functional unit of theinvention minus the central magnetic rod through the center hole (3)with fruste-conical edge (3). Black arrow representing negative pressurethrough center of functional unit is generated as a result of fluid flow(large white arrow) between upper contact (1) and lower base (4) plates.

FIG. 3: Conceptual drawing of smallest functional unit in an idealconfiguration with a eukaryotic cell (3) and its nucleus (1), sealingthe central hole (4) and the magnetizable rod (5) piercing the membraneof the cell (2) and magnetically capturing the nucleus (1). The baseplate (6) and magnetizable rod (5) may be physically connected andcomposed of the same suitable material to allow both to be selectivelyand electronically magnetizable.

FIG. 4: Close up view (dotted inset) of magnetized antibodies (3)binding to antigens on nuclei (2) and magnetized antibodies (3) in turnbeing attracted to the magnetized rod (4) with magnetic field turned on(filled in black).

FIG. 5: Close up view (dotted box inset) of antibody (2 and 4) bindingto antigens on nuclei (3) of a cell (1) and constant region of antibody(4) bound to antibodies immobilized (5) on the central rod (6) of theapparatus. (No magnetic antibody or magnetic attraction used for thisembodiment.)

FIG. 6: Close up view (dotted box inset) of antibody binding to antigenson nuclei (3) in a cell (1) with the constant region of antibody (6)bound to antibodies immobilized (4) on the magnetized rod (5) of theapparatus in addition to using magnetic antibodies (2) to magnetize thenucleus (3) and allow attraction to the magnetic rod (5). This approachuses direct antibody binding and magnetic attraction to magnetizablerod.

FIG. 7: Conceptual drawing of smallest functional unit after donor cellis pushed off of apparatus and donor nucleus (1) is still left behind onthe magnetized rod (5). The upper contact plate (2) has a central hole(4) through which a magnetizable rod (5) protrudes though and above thesurface plane of the upper contact plate (2) and the fruste-conical edge(3). The magnetizable rod (4) is physically attached to the base plate(6) and both are currently magnetized as illustrated by the black filledin appearance of the magnetizable rod (5).

FIG. 8: Conceptual drawing of the smallest functional unit of theapparatus being prepared for intracytoplasmic sperm injectionapplication. The upper contact plate (3) has a central hole (5) throughwhich a magnetizable rod (6) protrudes though and above the surfaceplane of the upper plate (3) and the fruste-conical edge (4). Themagnetizable rod (6) is physically attached to the base plate (7) andboth are currently magnetized as illustrated by the black filled inappearance of the magnetizable rod (5), which magnetically immobilizesthe magnetized antibody (2) binding to antigens on the sperm (1).

FIG. 9: Conceptual drawing of the smallest functional unit of theapparatus being used for intracytoplasmic sperm injection application.The upper contact plate (5) has a central hole (7) through which amagnetizable rod (8) protrudes though and above the surface plane of theupper contact plate (5) and the fruste-conical edge (6). Themagnetizable rod (8) is physically attached to the base plate (9) andboth are currently magnetized as illustrated by the black filled inappearance of the magnetizable rod (8), which magnetically immobilizesthe magnetized antibody (4), which is bound to antigens on the sperm(2). The recipient oocyte (1) with nucleus (3) rests atop the centralhole (7) and fruste-conical edge (6) and is impaled by the magnetizablerod (8).

FIG. 10: Conceptual drawing of the smallest functional unit of theapparatus being prepared for transfection of genetic agents (1), wherebya magnetically responsive (2) is linked to the genetic agent (1) and isattracted to the magnetized rod (6) which is connected to the base plate(7) and protrudes through the fruste-conical edge (3) of the centralhole (5), of the upper contact plate (4). Again, the magnetizable rod(6) and base plate (7) may be composed any suitable to allow amagnetizable surface which may be adjustable electronically.

FIG. 11: Conceptual drawing of the smallest functional unit of theapparatus being used for transfection of genetic agents, whereby amagnetically responsive element (4) is linked to the genetic agent(s)(2) and introduced into a cell (1) with a nucleus (3). The genetic agent(2) is attracted to the magnetized rod (8) which is connected to thebase plate (9) and protrudes through the fruste-conical edge (6) of thecentral hole (7), of the upper contact plate (5). Again, themagnetizable rod (8) and base plate (9) may be composed of any suitablematerial to allow a magnetizable surface which can be adjustableelectronically.

FIG. 12: Alternate conceptual drawing of the apparatus modified fortransfection of genetic agents (1), but in this case three times theamount of transfection may occur as there are 3 magnetizable rods (6),whereby a magnetically responsive label (2) is linked to the geneticagents (1) and is attracted to the magnetized magnetizable rod (6) whichis connected to the base plate (7) and protrudes through the,fruste-conical edge (4) of the central hole (5), of the upper contactplate (3). Again, the magnetizable rods (6) and base plate (7) may becomposed any suitable material to allow a magnetizable surface which maybe adjustable electronically.

FIG. 13: 3-fold transfection of genetic agents (3) into the recipientcell (1) with intact nucleus (2). Three magnetizable rods (4) arepictured in this representation to show semi-quantitative transfectionmay be achieved by modifying the number of rods (4) protruding throughthe central hole (5) of the apparatus.

FIG. 14: Alternate arrangement of apparatus for genetic agents (1)transfer using magnetized antibodies (2) to attract bound genetic agents(1) to magnetized magnetizable rod (3).

FIG. 15: An ideal three dimensional representation of the invention.Note magnetizable rods (3) fit through center (1) of upper contact plate(2). The lower base plate (4) is physically attached to the magnetizablerods (3) but a space between the upper contact plate (2) and lower lowerbase plate (4) exists to allow flow of tissue culture or buffer fluidbetween the plates.

FIG. 16: Longitudinal cross section representation of device. The uppercontact plate (7) is surrounded over the top surface with a ledge (1) tocreate a culture well space (2) to hold tissue culture media or bufferfluid. The magnetizable rods (3) are physically attached to the baseplate (6) and protrude through the central hole (4). Note the space (5)between each plate (6 and 7) to allow flow through of cell culturemedia/buffer.

FIG. 17: Sagittal, cross-section view of device. The upper contact plate(7) is surrounded over the top surface with a ledge (2) to create aculture well space (1) to hold tissue culture media or buffer fluid. Themagnetizable rods (4) protrude through the central hole (3) and isphysically attached to the base plate (6). Note the flow through port(5) in the space (diagonal hatching) between each plate (6 and 7) toallow flow through of cell culture media/buffer.

FIG. 18: Three dimensional view of invention with entry and exit flowthrough ports. Dark arrows signify direction of flow. Occlusion of thedistal flow through port (right side of drawing) results in flow out ofcentral holes of upper contact plate.

FURTHER EMBODIMENTS

While at least one method of use of the apparatus has been described, aperson of ordinary skill in the art can see that a variety of steps maybe used for the method and apparatus, and it is possible to add orremove optional steps to the method detailed.

1. A method of automated transfer of genetic agents in eukaryotic orprokaryotic cells at the microscopic and/or nanometric level, with themethod comprising: a. preparation of donor genetic material by: i.removal of genetic agent by first coating it with magneticallyresponsive labels prior to introduction into the device ii. introducingthe donor cells to the first reaction chamber ii. impaling the cell withan electronically magnetized rod with a vacuum at the base iii. removingthe rest of the donor cell by reversing the vacuum and washing away thecell into a waste chamber, but leaving the genetic agent magneticallybound to the rod iv. alternatively using no donor cell but introducinginto the first reaction chamber genetic agents with magneticallyresponsive labels b. preparation of the recipient cells using the samedevice but in a separate second reaction chamber by: i. removingredundant pre-existing genetic agents by coating it with 381magnetically responsive labels prior to introduction into the device ii.impaling the cell with an electronically magnetized rod with a vacuum atthe base and iv. removing the rest of the recipient cell by reversingthe vacuum and leaving the genetic agent magnetically bound to the rod.c. insertion of donor genetic agents into the recipient cells by: i.introducing the treated recipient cells from 1 b into the first reactionchamber of 1 a. i. impaling the recipient cell with the electronicallymagnetized rod holding the donor genetic agent by activating a vacuum atthe base and ii. releasing the genetic agent into the cytoplasm of thecell by electronically removing the magnetic field or reversing thepolarity of the rod and finally iii. releasing the cell by reversing thevacuum and allowing the newly transfected cell to roll free from theimpaling rod into a collection chamber.
 2. A device composed of: a.contact plate with raised edges to allow containment of cellmedia/buffer b. the same contact plate with a hole, with or withoutfruste-conical edges c. a second base plate beneath the upper contactplate d. a rod connected to the same base plate and protruding throughthe center of the contact plate hole past the plane of the contact platee. a space between the two plates to allow the flow of cell media/bufferwhich creates a vacuum affect through the contact plate holes in thedirection of the base plate f. an entry and exit flow port at thelongitudinal ends of the plates communicating and regulating the flow ofcell media/buffer g. the same exit port may be occluded to causepositive pressure out the central holes of the upper contact plate awayfrom the base plate and contact plate to push cells off the contactplate h. the same rod and base plate is electronically magnetizable andadjustable in field strength, polarity and/or neutrality i. the same rodmay be coated with magnetic or paramagnetic genetic agents whenmagnetized j. multiple reaction chambers with: i. entry and exitchannels leading to other reaction chambers or waste or collectionchambers ii. inter-chamber channels to cells and fluid to flow betweenthe chambers k. the same rod and hole which may be further arranged intoan array of multiple rod hole units but upon the same base plate andwithin the same reaction chamber l. fabrication being free of endotoxinm. dimensions to allow the entire apparatus to be housed within a Petrior tissue culture dish or any suitable tissue culture container suitablefor tissue culture n. dimensions to allow visualization under amicroscope
 3. Alternately the method and device may be composed of orutilize: a. a pleurality of rod and hole units to allow transfer and/orremoval of genetic agents from multiple cells simultaneously b.magnetized rods and/or base with a solid-state, pre-determined magneticpolarity and strength without the need or use of electricity c. apleurality of magnetizable rods within each hole to provide asemi-quantitative increase in transfer of genetic agents d. standardantibodies (without any magnetization) recognizing and binding to thegenetic agents and immobilized antibodies on the rod recognizing theconstant portion of the antibodies bound to the genetic agents e.translucent material to allow visualization under a standard or invertedmicroscope or scanning tunneling microscope or CCD or light sensingsurface f. flourochrome, fluorophore labels to visually detect geneticagents immobilized on the rods g. glass, plastic, silicone or anysuitable material to fulfill the functional criterion of the inventioni. manufacture by lithography, laser or chemical etching or any meansnecessary to fulfill functional criterion.